Abstract Body: Background: Hypertriglyceridemia is an atherosclerosis risk factor that is impacted by dietary triacylglyceride (TAG) absorption via the small intestine and endogenous hepatic production. Our laboratory reported that Dennd5b-deficient mice have impaired intestinal triglyceride-rich lipoprotein (TRL) secretion and resist diet-induced hepatic steatosis, hyperlipidemia, and atherosclerosis. Electron microscopy imaging of intestinal epithelium of Dennd5b^-/- mice revealed significant accumulation of intracellular chylomicron secretory vesicles, suggesting a post-Golgi secretion defect. Since DENND5B is expressed by both TRL-secreting organs (liver and small intestine) in Humans, we hypothesized that DENND5B disruption attenuates TRL secretion in both tissues.
Methods and Results: To determine if DENND5B disruption impairs TRL secretion in human cells, we generated DENND5B^-/- intestinal epithelial (Caco-2) and hepatocyte (HepG2) cell lines using CRISPR and verified lack of DENND5B protein via western blot. DENND5B disruption significantly reduced TAG secretion compared to DENND5B^+/+ controls (Caco-2: -67.8%, p<0.0001; HepG2: -91.0%, p<0.0001) without affecting cellular TAG content. To define the mechanism by which DENND5B influences TRL secretion, tagged DENND5B plasmids were transfected into HepG2 and Caco-2 cells for subcellular visualization and proximity-ligation based identification of DENND5B binding partners. Super-resolution microscopy of HepG2 cells revealed distinctive localization of DENND5B and APOB-positive puncta along microtubules. Caco-2 cell imaging shows similar APOB and DENND5B distributions. Immunoprecipitation of DENND5B-associated proteins in both cell types show motor protein-binding, membrane remodeling, and microtubule-membrane tethering protein enrichment, highlighting a potential role for DENND5B in vesicular trafficking or fusion with the plasma membrane.
Conclusions: These data support the hypothesis that DENND5B is involved in TRL secretion from human enterocytes and hepatocytes. Mechanistically, these studies associate DENND5B physically to the cytoskeleton and functionally to membrane docking and secretion. These findings also introduce new potential players in TRL exocytosis. Understanding the mechanistic details of this process may provide novel targets for regulation of plasma TAG and APOB concentrations.
This research was funded by the American Heart Association Pre-Doctoral Fellowship to Olivia Hage, and NIH grant R01DK133184.